Current Biology 19, 924–929, June 9, 2009 ª2009 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2009.04.018

ReportThe PCP Pathway Instructs the PlanarOrientation of Ciliated Cellsin the Xenopus Larval Skin

Brian Mitchell,1,3 Jennifer L. Stubbs,1 Fawn Huisman,2

Peter Taborek,2 Clare Yu,2 and Chris Kintner1,*1The Salk Institute for Biological StudiesLa Jolla, CA 92037USA2Department of Physics and AstronomyUniversity of California, IrvineIrvine, CA 92697USA

Summary

Planar cell polarity (PCP) is a property of epithelial tissueswhere cellular structures coordinately orient along a two-

dimensional plane lying orthogonal to the axis of apical-basal polarity [1]. PCP is particularly striking in tissues

where multiciliate cells generate a directed fluid flow, asseen, for example, in the ciliated epithelia lining the respira-

tory airways or the ventricles of the brain. To producedirected flow, ciliated cells orient along a common planar

axis in a direction set by tissue patterning, but how this isachieved in any ciliated epithelium is unknown [2]. Here,

we show that the planar orientation of Xenopus multiciliatecells is disrupted when components in the PCP-signaling

pathway are altered non-cell-autonomously. We also showthat wild-type ciliated cells located at a mutant clone border

reorient toward cells with low Vangl2 or high Frizzled activity

and away from those with high Vangl2 activity. These resultsindicate that the PCP pathway provides directional non-cell-

autonomous cues to orient ciliated cells as they differen-tiate, thus playing a critical role in establishing directed

ciliary flow.

Results and Discussion

Planar cell polarity (PCP) has been extensively studied inDrosophila, where it is evident in the ordered projection ofhairs on the wing and abdomen or in the orientation of omma-tidia in the eye [3]. The genes required to orient these struc-tures include those encoding the core components of thePCP-signaling pathway—namely three intracellular proteins,Prickle, Dishevelled, and Diego, and three transmembraneproteins, Flamingo, Frizzled, and Van Gogh (also known asStrabismus) (reviewed in [4–6]). In genetic mosaics, two ofthese genes, Frizzled and Van Gogh, produce profound non-cell-autonomous phenotypes in which the orientation ofwild-type cells adjacent to a mutant clone is redirected inwardor outward [6–8]. In addition, Frizzled and Van Gogh dynami-cally accumulate during PCP signaling at opposite sides ofa polarized cell (reviewed in [4]). These and other observationssuggest that Frizzled and Van Gogh, in combination withFlamingo, act as directional cues to align cells along a planar

*Correspondence: kintner@salk.edu3Present address: Department of Cell and Molecular Biology, Northwestern

University, Chicago, IL 60611, USA

axis based on local cell-to-cell comparisons [9–11]. PCPsignaling is also conserved in vertebrates. Homologs of severalPCP components are known to localize asymmetrically withinpolarized cells, such as cochlear hair cells, and to disruptPCP in several tissues when mutant (reviewed in [5, 12]).

The role of the PCP pathway in orienting ciliary flow has beenstudied in the Xenopus larval skin by using morpholinos toknock down the cytoplasmic PCP component, Dishevelled,or two downstream effectors of PCP called inturned and fuzzy[13, 14]. When all three Xenopus Dishevelled homologs are tar-geted by morpholinos (Dvl1-3), basal bodies (BBs) fail to dockat the apical surface and cilia are lost, a phenotype alsoobserved in morphants of inturned and fuzzy. Though thisphenotype is not a defect in PCP per se, it does suggest thatPCP components are required in a targeting mechanism thatlocalizes and docks BBs at the apical membrane where ciliaoutgrowth occurs. However, Dishevelled function can alsobe disrupted in embryos by expressing a well-characterized,dominant-negative mutant of Dvl2 called Xdd1 [14]. In theseembryos, cilia now form and beat but fail to polarize alonga planar axis, suggesting that Dishevelled also functionsdownstream of BB docking in a mechanism that establishestheir rotational orientation. Because the Dishevelled proteinshave functions outside of the PCP pathway [15, 16], it remainsunclear whether cell-cell interactions involving the PCPpathway are required to align ciliated cells along a planar axis.

To address the role of cell-cell interactions in orienting cili-ated cells, we exploited how these cells arise and arepatterned during Xenopus skin development [17]. Classicgrafting experiments in other amphibian species have shownthat the direction of ciliary flow along the anterior to posterioraxis (A-P) is set by a patterning event that occurs soon aftergastrulation and prior to ciliated cell differentiation [18, 19].At this stage, the developing skin in Xenopus embryos is notone epithelial layer as found in other amphibians but is twolayered, and the ciliated cells arise as precursors in the innerlayer before intercalating into the outer epithelial layer. Thus,if the global axis of planar polarity is also fixed this early inthe Xenopus skin, then ciliated cell precursors presumablyacquire an orientation when they intercalate, based on cuesestablished earlier in the outer epithelium. To confirm whenthe A-P polar axis is set in the skin of Xenopus laevis, werotated a small patch of developing skin before and aftergastrulation, allowed the embryos to develop, and scoredthe subsequent orientation of ciliated cells in the graft relativeto the host (Figure 1A). Cilia orientation was examined in thesegrafts functionally by flow measurements (Movies S1 and S2available online) and by measuring the rotational orientationof basal bodies with a confocal assay [14]. The confocal assaymeasures basal body orientation by using two fusion proteinsto label basal bodies with RFP and the rootlets with GFP (seeExperimental Procedures and Figure 2). The results show thatthe planar orientation of the skin is set in Xenopus soon aftergastrulation (Figure 1) and prior to ciliated cell differentiation,implying that the ciliated cells only acquire their planar orienta-tion when they later join the epithelium during intercalation.

To determine whether the PCP pathway is required in outercells to orient intercalating ciliated cells, we used a

PCP Signaling in Ciliated Cell Orientation925

transplantation assay to selectively disrupt the PCP pathwayin ciliated cells or in the epithelia into which they intercalate(Figure 2A). This assay was first used to determine whetherthe Dishevelled mutant Xdd1 disrupts the rotational axis ofBBs by acting solely in ciliated cells or whether it also disruptsthe ability of outer cells to orient ciliated cells non-cell-auton-omously [14]. When Xdd1-expressing ciliated cells intercalateinto wild-type outer cells, the polar orientation of the cilia isseverely disrupted, as predicted for a cell-autonomous pheno-type (Figures 2B and 2D). This disorientation was evident attwo levels: BB orientation was severely disorganized withincells (Figures 2B and 2D and short arrows in 2F), and themean cilia orientation of ciliated cells failed to converge alongthe A-P axis (mean direction of arrows in Figure 2F). Bycontrast, when wild-type ciliated cells intercalated into outerepithelium expressing Xdd1, their BBs oriented normallywithin cells (Figures 2C and 2E and long arrows inFigure 2G), and ciliated cells were polarized in a posterior

Figure 1. Timing of Planar Axis Determination

(A) Diagram of a grafting experiment in which both layers of the developing

skin were isolated at the indicated stage, rotated 180�, and transplanted ho-

motopically onto host embryos. At stage 28, the orientation of ciliated cells

both inside and outside the graft was determined by confocal microscopy

(see Experimental Procedures and Figure 2).

(B–E) Ciliated cell orientation following a stage 10 (B and C) or stage 16

(D and E) skin transplant and scored outside (B and D) and inside of the

transplant (C and E). Each arrow represents the mean orientation of BBs

within a cell, and arrow length represents the complement of circular

variance around that mean. Colors represent data from separate embryos.

Ciliated cells normally orient posteriorly with a ventral bias.

direction with a ventral bias as normal (mean direction ofarrows in Figure 2G compared to Figures 1B and 1D). Thus,Xdd1 disrupts BB orientation cell autonomously but cannotdisrupt ciliated cell polar orientation in a noncell-autonomousfashion.

Figure 2. Xdd1 Functions Cell Autonomously to Disrupt Cilia Orientation

(A) Diagram of the assay in which outer cells are grafted from a donor onto

a host embryo prior to gastrulation.

(B and C) Confocal image of an Xdd1-expressing ciliated cell surrounded by

a transplant of wild-type outer cells (B) or of wild-type ciliated cells sur-

rounded by a transplant of Xdd1-expressing outer cells (C). Ciliated cells

express a Centrin-RFP (red) fusion protein that labels BBs and a CLAMP-

GFP (green) fusion protein that labels the rootlet.

(D and E) The areas indicated in (B) and (C) are magnified 53 in (D) and (E),

respectively. Arrows indicate the direction of cilium orientation based on

centrin and CLAMP staining.

(F and G) Circular graphs depicting mean cilia orientation of cells from

a transplant of wild-type outer cells onto Xdd1-injected ciliated cells (D) or

Xdd1-injected outer cells transplanted onto wild-type ciliated cells (E).

Each arrow represents the mean direction of BBs within a cell, and the

length represents one minus the circular variance around the mean. There-

fore, cells with short arrows vary more in BB orientation than those with long

arrows. In wild-type embryos, ciliated cells beat in a posterior direction with

a ventral bias.

Current Biology Vol 19 No 11926

We next asked whether the transmembrane components ofthe PCP pathway are required to orient ciliated cells, initially bytargeting a Xenopus homolog of Van Gogh called Vangl2, byusing a morpholino designed to block the translation of Vangl2RNA (Vangl2MO). The ability of the Vangl2MO to disrupt Vangl2function was first tested in the mesoderm, where PCPsignaling in general and Vangl2 in particular is required forthe polarized cell movements that underlie axial elongation[20]. As predicted, injecting Vangl2MO, but not a control MO,into the marginal zone of two-cell embryos produced strongdefects in axial elongation (Figure S1). We then asked whetherdisrupting Vangl2 function in the skin by injecting the Vangl2MO

into the animal pole of two-cell embryos causes defects in cil-iogenesis, as reported previously for a knockdown of Dvl1-3,Inturned, or Fuzzy [13, 14]. Indeed, in Vangl2MO morphants,BBs failed to dock at the apical surface, and cilia were dramat-ically reduced in number. This phenotype was substantiallyrescued by coinjecting a synthetic Vangl2 mRNA lackingsequences targeted by the morpholino (Figure S2). Althoughfewer in number, the extant cilia in Vangl2 morphants havea similar beat frequency to that of wild-type cilia but are disor-ganized in orientation (Figure S3 and Movies S3 and S4). Thus,these results suggest that the PCP pathway and, specifically,Vangl2 is required for BB apical localization and ciliogenesisbut is unlikely to be required for cilia motility [13, 14].

Because the deleterious effects of Vangl2 on ciliogenesisare likely to be cell autonomous, as shown above for Xdd1,we next asked whether a loss of Vangl2 function in outer cellsresulted in non-cell-autonomous effects on the polarity ofwild-type ciliated cells (Figure 3A). As a control, ciliated cellorientation was found to be normal when a wild-type outerlayer (mRFP) was transplanted onto a wild-type embryo(Figure 3, compare [B] and [C]). By contrast, when outer layercells were transplanted from Vangl2MO-injected donors ontowild-type hosts, the orientation of the intercalated wild-typeciliated cells within the clone was severely disorganizedcompared with cells outside the clone (Figure 3, compare [E]and [D]). Significantly, the non-cell-autonomous disruption ofcilia orientation obtained in this experiment was distinct fromthat obtained cell autonomously with Xdd1 above. Specifi-cally, the Vangl2 mutant outer cells did not disrupt the orienta-tion of BBs within ciliated cells or the coordinated beating ofcilia (longer arrows in Figure 3E; compare Movie S2 to S3)but, rather, the orientation of ciliated cells along the A-P axis.Indeed, ciliated cell orientation varied significantly more onaverage around a mean direction within a Vangl2MO-injectedclone compared to a control (p = 0.000471). These resultssuggest that ciliated cells acquire their orientation via cell-cell interactions with the outer layer, as they intercalate andsuggest that the orientation cue requires Vangl2.

In cases in which PCP signaling has been shown to act,overexpression of components of the PCP pathway in gain-of-function experiments often causes similar polarity defectsas those observed in loss-of-function experiments. Thus, tofurther assess the role of the PCP pathway in outer cells, wetransplanted outer cells overexpressing Vangl2 RNA (Van-gl2OE) onto wild-type hosts. Ciliated cell orientation withinthese Vangl2OE clones was also disrupted (Figure 3, compare[F] and [G]), varying significantly more around a mean direc-tion compared to controls (p = 0.0075). Thus, both the loss-and gain-of-function experiments with Vangl2 support theidea that outer cells provide cues to orient ciliated cells andthat the proper levels of Vangl2 are required to generatethis cue.

In Drosophila, changing the activity of Frizzled in mutantclones also has profound noncell-autonomous effects onneighboring wild-type cells [1]. However, vertebrates have

Figure 3. Ciliated Cell Orientation Is Disrupted Non-Cell-Autonomously by

Changes in Vangl2 and Fz3 Activity

(A) Diagram of an assay in which outer cells from donor embryos injected

with Vangl2MO, Vangl2, Fz3, or RFP RNA were grafted onto wild-type host

embryos. Ciliated cell orientation was measured with confocal microscopy

(see Experimental Procedures and Figure 2) either outside of the clone (>30

cell diameters from the clone) or inside of the clone.

(B–I) Circular graphs of ciliated cell orientation for wild-type cells outside of

the clones (B, D, F, and H) and for wild-type cells surrounded by trans-

planted outer cells injected with mRFP (D), Vangl2MO (E), Vangl2 OE (G), or

Fz3 OE (I).

PCP Signaling in Ciliated Cell Orientation927

a large number of Frizzled homologs, in contrast to Vangl2,and it is not clear which and how many of these might berequired for PCP signaling in the skin [5, 12]. Therefore, wefocused on the overexpression of Frizzled-3 (Fz3OE) becausemice mutant for Fz3, when combined with those in Fz6, havedefects in axial elongation and polar orientation defects inhair cells [21]. When wild-type ciliated cells intercalate intoouter cells that overexpress Fz3, their orientation is also dis-rupted, showing more variation around a mean directioncompared to control (p = 0.00021) (Figure 3, compare [I]and [H]). Thus, these results suggest that changes in the levelsof PCP signaling alter ciliated cell orientation non-cell-autonomously.

One interpretation of the results above is that outer cellsrequire the proper levels of Vangl2 and Frizzled activity,perhaps indirectly, to generate an orientation cue for ciliatedcells. Alternatively, Vangl2 and Frizzled may be acting asthey do in PCP signaling in Drosophila by instructively polar-izing neighboring ciliated cells. The key observations thatdistinguish between these two possibilities in Drosophila arethe different directional nonautonomous phenotypes thatoccur at clone boundaries mutant for Frizzled and Van Gogh[7, 8]. Thus, to determine whether Vangl2 is also acting instruc-tively in outer cells to orient ciliated cells, we analyzed theorientation of ciliated cells lying at the anterior and posteriorboundary of an outer-cell transplant (Figure 4A and S4).

Ciliated cells located at the anterior and posterior borders ofa clone of wild-type cells (mRFP) orient their cilia in the normalposterior direction with an w45� ventral bias (Figures 4B, 4C,S5A, and S5B). Ciliated cells located at the anterior border ofVangl2MO clones are still oriented posteriorly but with a lesspronounced ventral bias (Figures 4D and S5C). In starkcontrast, ciliated cells at posterior border of Vangl2MO clonesare significantly reversed, on average, 124� (p = 7.57E-5) rela-tive to control cells and 166� relative to the cells at the anteriorborder (Figures 4E and S5D). We see a reciprocal effect on theorientation of ciliated cells at the border of Vangl2OE clones(Figures 4F and 4G). Ciliated cells at the anterior border of Van-gl2OE clones (Figures 4F and S5E) are reversed 154� relative tocontrols (p = 1.81E-18), whereas those at the posterior border(Figures 4G and S5F) have lost their ventral bias and, thus,shifted 48� relative to the control. In Drosophila, Van Goghand Frizzled have reciprocal effects on orienting cells at cloneboundaries [6–8]. Though less striking than the Vangl2 results,the ciliated cells located at the anterior border of Fz3OE clones(Figure 4H) are shifted 37� relative to controls (p = 9.7E-7) butare still oriented in a posterior direction. Ciliated cells at theposterior borders, however, are reversed 71� relative to thecontrols in an anterior direction (p = 6.03E-7) and are, onaverage, shifted 108� relative to the cells at the anterior border.These results provide strong evidence that Vangl2 and Frizzledlevels in outer cells instructively orient the planar polarity ofciliated cells but in opposite directions: ciliated cells orientthe beating of their cilia toward outer cells with lower levelsof Vangl2 activity and to those with higher levels of Frizzled.

These data support a model in which ciliated cells arepatterned along the A-P axis of the developing skin based oncues that they encounter when they intercalate into the outerepithelium. Moreover, our data suggest that PCP signalingacts as a major cue in this patterning event whereby Vangl2and Fz3 (or related Frizzled[s]) instruct the polar orientationof intercalating cells in a reciprocal manner. Therefore, theinstructive signaling cues that we observe in our transplantassay provide strong functional evidence that ciliated cells

are oriented by local asymmetry in PCP activity and representthe first direct evidence for directional noncell autonomyduring PCP signaling in a vertebrate system. PCP signaling

Figure 4. Vangl2 and Fz3 Have Directional, Non-Cell-Autonomous Effects

on Ciliated Cell Orientation

(A) Diagram of an assay in which wild-type ciliated cells are located at the

anterior and posterior border of an outer-cell clone from donor embryos in-

jected with Vangl2MO, Vangl2, Fz3, or RFP RNA. Ciliated cell orientation at

the clone border was measured with confocal microscopy (Figure S4).

(B–I) Circular graphs of ciliated cell orientation for cells located at the ante-

rior border (B, D, F, H) or for those located at the posterior border (C, E, G, I)

under the indicated experimental conditions. Different colors represent data

from different experiments.

Current Biology Vol 19 No 11928

in Drosophila is accompanied by and potentially attributed tothe asymmetric localization of Frizzled and Van Gogh alongthe planar axis to opposite sides of each cell (reviewed in[4]). Accordingly, our functional data would predict that Vangl2and Frizzled activity are differentially restricted to the posteriorand anterior sides of skin cells, respectively. It remains to bedetermined whether this differential activity involves asym-metrical protein localization, as proposed in Drosophila, oranother mechanism.

In contrast to Vangl2 and Fz3, the Dvl2 mutant Xdd1 did notcause a disorientation of ciliated cells when expressed inclones of outer cells, even though, in the converse experiment,it disrupted the orientation of BBs in a cell-autonomousmanner. This result is reminiscent of findings in Drosophila,suggesting that Dishevelled is not required to generate orpropagate intercellular PCP signaling, at least over clonedistances, but is only required intracellularly for cells topolarize [22, 23]. Disrupting Dishevelled activity in the outercells by using additional approaches will be required to fullyaddress the role of Dishevelled in PCP signal propagationboth intercellularly and intracellularly.

Our data also show that BBs fail to dock apically, and cilio-genesis fails in Vangl2 morphants, as reported previously forDvl1-3, Inturned, and Fuzzy [13, 14]. Intriguingly, mammalianVangl2 has been reported on vesicular structures that localizeto the BB in human respiratory ciliated cells [24]. Our data,therefore, add further support to the idea that BBs are posi-tioned apically in ciliated cells by vesicular targeting eventsinvolving multiple components of the PCP pathway [14]. Dueto the ciliogenesis defects, we cannot test whether Vangl2also has a cell-autonomous role in establishing the rotationalorientation of individual BBs. Nonetheless, our observationssuggest that, at the same time PCP components positionBBs apically, they are also used to orient ciliated cellsalong the planar axis via interactions with cells in the outerepithelium.

Experimental Procedures

Transplant Assays and Explant Cultures

Xenopus laevis embryos were obtained by in vitro fertilization with standard

protocols [25]. To mark transplanted tissue, we injected embryos four times

at the two- to four-cell stages with capped, synthetic mRNA-encoding [25]

membrane-localized form of RFP (mRFP). At stage 10, a fine needle or hair

was used to peel off the outer layer from a region of the ectoderm from

a donor embryo, which was transferred onto the host embryos after

removing a similar patch of outer cells. While the transplanted tissue healed

onto the host embryo, it was kept in place by pressing down with a small

piece of a glass coverslip, held in place with silicone grease. In experimental

transplants, host embryos were not only injected with mRFP but also with

a Vangl2 MO (50-ACTGGGAATCGTTGTCCATGTTTC-30, Gene Tools),

Control MO (50-CTAGCGCTGTAAGGAGCCATCCTGT-30), Vangl2 [26], or

Fz3 RNA [27]. Transplants were performed in Danilchik’s buffer + 0.1%

BSA [28]. After healing of the transplanted tissue, we returned embryos to

0.1 3 Marc’s Modified Ringers (MMR) [25] until stage 28 when they fixed

overnight on ice in 4.0% paraformaldehyde in phosphate-buffered saline

(PBS). After mounting, tadpoles were imaged with a BioRad Radiance

2100 confocal mounted to a Zeiss inverted microscope with a 633 objec-

tive. Grafted tissues were identified based on the RFP tracer and were

analyzed when localized to the middle flank. Ciliated cells were imaged

that were either within the grafted tissues, at least 30 cell diameters outside

the grafted tissue, or located at the anterior or posterior border and touching

both grafted and host cells.

Confocal Assay for Cilia Orientation

To score ciliated cell orientation, we determined cilia direction along the

polar axis by measuring BB orientation with confocal microscopy [14].

This assay involves expressing two fusion proteins in host embryos. One

protein, called CLAMP, is fused to GFP and localizes to the striated rootlet,

a structure that marks the rotational axis of the BB by projecting in the

opposite direction of ciliary beating. The second protein, Centrin2, is fused

to RFP and localizes to the basal body. When expressed in ciliated cells with

RNA injection, the two fusion proteins decorate the BB and rootlet such that

orientation can be easily scored by confocal microscopy. The orientation of

w100 BBs was scored per cell and used to calculate the mean orientation of

cilia within a cell as a measure of a cell’s overall planar polarity, wherein the

mean direction of a cell is denoted as an arrow on a circular graph and the

length of the arrow represents the variance around that mean [29].

Immunostaining and Confocal Microscopy

Ciliated cells were immunostained by fixing embryos with 4.0% paraformal-

dehyde in PBS for 1 hr on ice. Tissue was stained by overnight incubation in

rabbit anti-ZO-1 (Zymed 1:200) and mouse monoclonal anti-acetylated

tubulin (Sigma 1:1000) or anti-g tubulin GTU88 (Sigma 1: 500) primary anti-

bodies and by a 4 to 6 hr incubation in anti-rabbit Cy3 and anti-mouse

Cy2 secondary antibodies (Jackson Immunoresearch). Antibody incuba-

tions were performed with PBS containing 0.1% Triton X-100 and 10%

heat-inactivated normal goat serum and were washed with PBS containing

0.1% Triton X-100. Basal bodies and rootlets were labeled by injecting

synthetic messenger RNA encoding centrin2-RFP and Clamp-GFP fused

at the carboxyl terminus as previously described [14]. After mounting,

embryos were imaged on a BioRad Radiance 2100 confocal mounted to

a Zeiss inverted microscope with a 633 objective. Movies of cilia beating

were taken at 6688 fps with a Vision Research Phantom 7.2 mounted to

an Olympus BX51 microscope with a 1003 objective.

Data Analysis

Basal body-rootlet orientation was scored with Matlab, and statistical anal-

ysis and circular plotting were done with Oriana 2.0 (Kovach Computing

Services) circular statistics software. Each arrow on the polar plot repre-

sents the orientation of a single ciliated cell based on scoring the orientation

of, on average, 100 cilia (basal body-rootlets) per cell. Experimental values

were compared to control values with a two-tailed Student’s t test.

Supplemental Data

Supplemental Data include five figures and five movies and can be found

with this article onlineat http://www.cell.com/current-biology/supplemental/

S0960-9822(09)00977-4.

Acknowledgments

The authors thank members of the Kintner lab for comments on the manu-

script. This work was supported in part by the UCI Center for Complex

Systems Biology under NIH P50GM076516; by the UCI Institute for Surface

and Interface Science; by an NIH grant GM076507 to C.K.; and by a Parker B.

Francis fellowship to B.M.

Received: October 7, 2008

Revised: March 5, 2009

Accepted: April 2, 2009

Published online: May 7, 2009

References

1. Zallen, J.A. (2007). Planar polarity and tissue morphogenesis. Cell 129,

1051–1063.

2. Marshall, W.F., and Kintner, C. (2008). Cilia orientation and the fluid

mechanics of development. Curr. Opin. Cell Biol. 20, 48–52.

3. Adler, P.N. (2002). Planar signaling and morphogenesis in Drosophila.

Dev. Cell 2, 525–535.

4. Strutt, D. (2008). The planar polarity pathway. Curr. Biol. 18, R898–R902.

5. Seifert, J.R., and Mlodzik, M. (2007). Frizzled/PCP signalling: A

conserved mechanism regulating cell polarity and directed motility.

Nat. Rev. Genet. 8, 126–138.

6. Lawrence, P.A., Struhl, G., and Casal, J. (2007). Planar cell polarity: One

or two pathways? Nat. Rev. Genet. 8, 555–563.

7. Vinson, C.R., and Adler, P.N. (1987). Directional non-cell autonomy and

the transmission of polarity information by the frizzled gene of

Drosophila. Nature 329, 549–551.

8. Taylor, J., Abramova, N., Charlton, J., and Adler, P.N. (1998). Van Gogh:

A new Drosophila tissue polarity gene. Genetics 150, 199–210.

PCP Signaling in Ciliated Cell Orientation929

9. Wu, J., and Mlodzik, M. (2008). The frizzled extracellular domain is

a ligand for Van Gogh/Stbm during nonautonomous planar cell polarity

signaling. Dev. Cell 15, 462–469.

10. Chen, W.S., Antic, D., Matis, M., Logan, C.Y., Povelones, M., Anderson,

G.A., Nusse, R., and Axelrod, J.D. (2008). Asymmetric homotypic inter-

actions of the atypical cadherin flamingo mediate intercellular polarity

signaling. Cell 133, 1093–1105.

11. Strutt, H., and Strutt, D. (2008). Differential stability of flamingo protein

complexes underlies the establishment of planar polarity. Curr. Biol.

18, 1555–1564.

12. Wang, Y., and Nathans, J. (2007). Tissue/planar cell polarity in verte-

brates: New insights and new questions. Development 134, 647–658.

13. Park, T.J., Haigo, S.L., and Wallingford, J.B. (2006). Ciliogenesis defects

in embryos lacking inturned or fuzzy function are associated with failure

of planar cell polarity and Hedgehog signaling. Nat. Genet. 38, 303–311.

14. Park, T.J., Mitchell, B.J., Abitua, P.B., Kintner, C., and Wallingford, J.B.

(2008). Dishevelled controls apical docking and planar polarization of

basal bodies in ciliated epithelial cells. Nat. Genet. 40, 871–879.

15. Wallingford, J.B., and Habas, R. (2005). The developmental biology of

Dishevelled: An enigmatic protein governing cell fate and cell polarity.

Development 132, 4421–4436.

16. Boutros, M., and Mlodzik, M. (1999). Dishevelled: At the crossroads of

divergent intracellular signaling pathways. Mech. Dev. 83, 27–37.

17. Stubbs, J.L., Davidson, L., Keller, R., and Kintner, C. (2006). Radial inter-

calation of ciliated cells during Xenopus skin development. Develop-

ment 133, 2507–2515.

18. Twitty, V.C. (1928). Experimental studies on the ciliary action of

amphibian embryos. J. Exp. Zool. 50, 310–344.

19. Tung, T.C., and Tung, Y.-F.Y. (1940). Experimental studies on the deter-

mination of polarity of ciliary action in anuran embryos. Arch. Biol.

(Liege) 51, 203–218.

20. Heisenberg, C.P., and Tada, M. (2002). Wnt signalling: A moving picture

emerges from van gogh. Curr. Biol. 12, R126–R128.

21. Wang, Y., Guo, N., and Nathans, J. (2006). The role of Frizzled3 and

Frizzled6 in neural tube closure and in the planar polarity of inner-ear

sensory hair cells. J. Neurosci. 26, 2147–2156.

22. Strutt, D., and Strutt, H. (2007). Differential activities of the core planar

polarity proteins during Drosophila wing patterning. Dev. Biol. 302,

181–194.

23. Amonlirdviman, K., Khare, N.A., Tree, D.R., Chen, W.S., Axelrod, J.D.,

and Tomlin, C.J. (2005). Mathematical modeling of planar cell polarity

to understand domineering nonautonomy. Science 307, 423–426.

24. Ross, A.J., May-Simera, H., Eichers, E.R., Kai, M., Hill, J., Jagger, D.J.,

Leitch, C.C., Chapple, J.P., Munro, P.M., Fisher, S., et al. (2005). Disrup-

tion of Bardet-Biedl syndrome ciliary proteins perturbs planar cell

polarity in vertebrates. Nat. Genet. 37, 1135–1140.

25. Sive, H., Grainger, R.M., and Harland, R.M. (1998). The early develop-

ment of Xenopus laevis: A laboratory manual (Plainview, NY: Cold

Spring Harbor Press).

26. Goto, T., and Keller, R. (2002). The planar cell polarity gene strabismus

regulates convergence and extension and neural fold closure in Xeno-

pus. Dev. Biol. 247, 165–181.

27. Rasmussen, J.T., Deardorff, M.A., Tan, C., Rao, M.S., Klein, P.S., and

Vetter, M.L. (2001). Regulation of eye development by frizzled signaling

in Xenopus. Proc. Natl. Acad. Sci. USA 98, 3861–3866.

28. Davidson, L.A., Hoffstrom, B.G., Keller, R., and DeSimone, D.W. (2002).

Mesendoderm extension and mantle closure in Xenopus laevis gastru-

lation: Combined roles for integrin alpha(5)beta(1), fibronectin, and

tissue geometry. Dev. Biol. 242, 109–129.

29. Mitchell, B., Jacobs, R., Li, J., Chien, S., and Kintner, C. (2007). A posi-

tive feedback mechanism governs the polarity and motion of motile

cilia. Nature 447, 97–101.